Accumulating evidence indicates that nitric oxide (NO), through S-nitrosylation of Cys residues within multiple, functionally interrelated signaling elements, regulates agonist-induced desensitization and internalization of B- adrenergic receptors (B-ARs). We have demonstrated that NO synthases and the endogenous S-nitrosothiol (SNO), S-nitrosoglutathione, preserve cardiac contractility and prevent down-regulation of B-ARs during maintained agonist stimulation. These effects of NO/SNO on B-AR signaling and trafficking, which can be recapitulated in cellular systems, appear to be mediated, in significant part, by S-nitrosylation of the G protein-coupled receptor (GPCR) kinase, GRK2. Additional components of the B-AR system are also regulated by S-nitrosylation, which suggests a broad functional role for NO/SNO, exerted through targeted S-nitrosylation. In particular, we have recently identified the B-arrestins (Barr1 and Barr2) as targets of B-AR-coupled S-nitrosylation by NO synthases (eNOS and nNOS). S-nitrosylation of Barr2 by eNOS at a single critical site (Cys 410) regulates its protein-protein interactions with clathrin and adapter protein-2 (AP-2) in vitro and in vivo, thereby promoting agonist-mediated B{2}-AR internalization. However, differential regulation of Barr1 and Barr2 by S-nitrosylation and the consequences of these modifications for cardiac function, have not been explored. Our central hypothesis is that S-nitrosylation of the B-arrestins will provide a basis for control by NO of B-AR trafficking and signaling, with important ramifications in healthy and failing hearts. Moreover, we predict that the regulation of Barr1- and Barr2-specific interactomes by S-nitrosylation will provide a principal mechanism through which NO exerts its regulatory influence. We will carry out the following specific aims: Specific Aim 1. Elucidate the sites of agonist-dependent S-nitrosylation of the B-arrestins by eNOS and nNOS in cells and tissues. Specific Aim 2. Elucidate the consequences of B-arrestin S-nitrosylation for B-AR internalization and desensitization. Specific Aim 3. Assess the consequences of B-arrestin S-nitrosylation for p-AR-dependent signaling. Specific Aim 4. Assess the functional roles of B-arrestin S-nitrosylation in the intact heart. Collectively, these studies should provide fundamental and novel insights into B-AR regulation by NO in both healthy and failing hearts and may open a new area of research.